Bottom Line:
Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo.We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins.A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain.

ABSTRACTTDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo. Analysis of RNA binding by TDP-43 in brains from subjects with FTLD revealed that the greatest increases in binding were to the MALAT1 and NEAT1 noncoding RNAs. We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain.

Figure 1: Comparison of TDP-43 RNA binding in healthy and FTLD-TDP brain(a) To validate specificity of TDP-43 antibody for iCLIP, the 32P-labelled RNA bound to TDP-43 gel was isolated from control (Ctr) or knockdown (KD) HeLa cells in the presence or absence of UV crosslinking or anti-TDP-43 antibody. High and low RNase concentrations were used to confirm the presence of RNA bound to TDP-43. The arrows mark the positions on the gel corresponding to the size of TDP-43 monomer or dimer. TDP-43 Western analysis of input extracts confirmed TDP-43 knockdown, and GAPDH was used as a loading control. The full image is shown in Supplementary Fig. 1a(b) The proportion of cDNAs (out of all cDNAs that mapped to human genome) from the TDP-43 iCLIP experiments in the four types of samples that mapped to different RNA regions. (c) The proportion of cDNAs that mapped to different types of ncRNAs. (d) The proportion of cDNAs that mapped to individual RNAs with at least 10 cDNAs in any experiment. The RNAs with the largest significant change between control and FTLD-TDP brain (difference in proportion of cDNAs > 0.1% and p value < 0.05 by Student’s t-test, one-tailed, unequal variance) are marked in red. The long ncRNA MEG3 (maternally expressed 3) with the largest decrease in TDP-43 binding in FTLD-TDP is also marked. (e) Real-time PCR analysis of transcripts with largest TDP-43 iCLIP changes in total RNA isolated from control and FTLD-TDP brain samples. (f) iCLIP cDNA counts for TDP-43 crosslink positions in the NEAT1 gene in experiments from SH-SY5Y and hES cell lines, and healthy and FTLD-TDP tissue from different individuals. The blue bars represent sequences on the sense strand of the genome, and the height of the bars corresponds to the cDNA count. The positions of significant crosslink clusters (XL cluster) are shown on top, and the RNA sequence underlying the two man clusters is shown below, with UG repeats in pink. The graph shows the average proportion of cDNAs that map to NEAT1 transcript in each experiment (out of all cDNAs maping to the human genome), as well as standard deviation between experiments from different individuals. (g) iCLIP cDNA counts for TDP-43 crosslink positions in the SLC1A2 gene. The orange bars represent sequences on the antisense strand of the genome. The graph shows the average proportion of cDNAs that map to SLC1A2 3′ UTR in different experiments, as well as standard deviation between experiments from different individuals. The RNA sequence underlying the peak crosslinking sites is shown below, with UG repeats in pink.

Mentions:
TDP-43 in complex with its direct RNA targets was purified from UV-crosslinked cells and analysed on SDS-PAGE gel. The size of the dominant TDP-43-RNA complex present in the low and high RNase conditions corresponded to a single TDP-43 molecule bound to the RNA, however a complex corresponding to two TDP-43 molecules bound to the RNA was also present, particularly in the low RNase experiment (Fig. 1a). This result agreed with the finding of a past study that TDP-43 binds RNA as a homodimer2.

Figure 1: Comparison of TDP-43 RNA binding in healthy and FTLD-TDP brain(a) To validate specificity of TDP-43 antibody for iCLIP, the 32P-labelled RNA bound to TDP-43 gel was isolated from control (Ctr) or knockdown (KD) HeLa cells in the presence or absence of UV crosslinking or anti-TDP-43 antibody. High and low RNase concentrations were used to confirm the presence of RNA bound to TDP-43. The arrows mark the positions on the gel corresponding to the size of TDP-43 monomer or dimer. TDP-43 Western analysis of input extracts confirmed TDP-43 knockdown, and GAPDH was used as a loading control. The full image is shown in Supplementary Fig. 1a(b) The proportion of cDNAs (out of all cDNAs that mapped to human genome) from the TDP-43 iCLIP experiments in the four types of samples that mapped to different RNA regions. (c) The proportion of cDNAs that mapped to different types of ncRNAs. (d) The proportion of cDNAs that mapped to individual RNAs with at least 10 cDNAs in any experiment. The RNAs with the largest significant change between control and FTLD-TDP brain (difference in proportion of cDNAs > 0.1% and p value < 0.05 by Student’s t-test, one-tailed, unequal variance) are marked in red. The long ncRNA MEG3 (maternally expressed 3) with the largest decrease in TDP-43 binding in FTLD-TDP is also marked. (e) Real-time PCR analysis of transcripts with largest TDP-43 iCLIP changes in total RNA isolated from control and FTLD-TDP brain samples. (f) iCLIP cDNA counts for TDP-43 crosslink positions in the NEAT1 gene in experiments from SH-SY5Y and hES cell lines, and healthy and FTLD-TDP tissue from different individuals. The blue bars represent sequences on the sense strand of the genome, and the height of the bars corresponds to the cDNA count. The positions of significant crosslink clusters (XL cluster) are shown on top, and the RNA sequence underlying the two man clusters is shown below, with UG repeats in pink. The graph shows the average proportion of cDNAs that map to NEAT1 transcript in each experiment (out of all cDNAs maping to the human genome), as well as standard deviation between experiments from different individuals. (g) iCLIP cDNA counts for TDP-43 crosslink positions in the SLC1A2 gene. The orange bars represent sequences on the antisense strand of the genome. The graph shows the average proportion of cDNAs that map to SLC1A2 3′ UTR in different experiments, as well as standard deviation between experiments from different individuals. The RNA sequence underlying the peak crosslinking sites is shown below, with UG repeats in pink.

Mentions:
TDP-43 in complex with its direct RNA targets was purified from UV-crosslinked cells and analysed on SDS-PAGE gel. The size of the dominant TDP-43-RNA complex present in the low and high RNase conditions corresponded to a single TDP-43 molecule bound to the RNA, however a complex corresponding to two TDP-43 molecules bound to the RNA was also present, particularly in the low RNase experiment (Fig. 1a). This result agreed with the finding of a past study that TDP-43 binds RNA as a homodimer2.

Bottom Line:
Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo.We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins.A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain.

ABSTRACTTDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo. Analysis of RNA binding by TDP-43 in brains from subjects with FTLD revealed that the greatest increases in binding were to the MALAT1 and NEAT1 noncoding RNAs. We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain.